We have demonstrated that human cardiac stem cells (hCSC) derived from end-stage heart failure (ESHF) hearts are markedly superior in improving post-infarct LV function in comparison to age-match control hCSC. The goal of this application is to characterize the molecular mechanisms underlying this improved performance of ESHF-derived hCSC and to optimize their harvest, molecular characterization and application in a clinically-relevant model of post-MI cardiac failure. Improvement of LV function in ESHF patients, particularly children with end- stage heart failure, via stem cell therapy is of enormous importance since heart transplantation is the only other viable option and in limited supply. Despite their encouraging clinical Phase I results, cardiosphere derived cells (CDCs), comprised of heterogeneous cell types, including c- kit+ cells, are still not well characterized1. Our preliminary data shows that ESHF-derived hCDCs isolated from the left atrium improve ischemic left ventricular function better than ESHF-derived hCDCs from the right atrium, but whether different functional activity is present in all other heart chamber-derived hCDCs is still unknown. Furthermore, since we document significantly higher numbers of c-kit+ cells within ESHF-derived CDCs, the frequency of the c-kit+ cells within CDCs may be critical to recovery of function. Finally, ESHF-derived CDCs secrete higher levels of angiogenic cytokines that correlates with increased angiogenesis in the infarcted myocardium and higher levels of HIF-1a, but the mechanism for the increased cytokine secretion is unclear. Our hypothesis is that the effect of ESHF-derived hCDCs in improving myocardial function is dependent on the anatomic site of hCDC origin and molecular mechanisms by c-kit+ and HIF- 1a. These studies will clarify the biology and function of hCDCs by determining: 1) chamber specific differences amongst hCDCs with the potential need to modify for a more powerful myocardial functional activity, 2) the effect of the frequency of c-kit+ cells on LV recovery, and lastly, 3) the role of HIF-1a as a master cytokine regulator of the myocardial function of ESHF- derived hCDCs. ESHF patients, particularly children, are potentially the most to benefit from hCDC based therapies. This application is the first study designed to determine critical characteristics of ESHF-derived hCDCs and to uncover new mechanisms of their functional activity in a manner that may eventually influence future therapeutic interventions.